All studied vertebrate DNAs contain approximately 107 -108 residues of 5-methylcytosine (m5C) per haploid genome but the functional significance of this DNA methylation is poorly understood. The tissue-specificity of DNA methylation patterns and numerous studies of relationships between DNA methylation and transcription suggest that some m5C residues are specifically involved in repressing gene expression. Our newly discovered human placental protein, methylated DNA-binding protein (MDBP), might direct changes in transcription in response to DNA methylation. MDBP is, currently, the only known eukaryotic protein which binds specifically to DNA sequences containing m5C. MDBP-specific DNA sequences have been examined to date only from in vitro-methylated bacteriophage and plasmid DNAs because of their enormously lower sequence complexity than that of human DNA. The detailed data already obtained about prokaryotic MDBP sites serve as a background for the following experiments. In the proposed research, MDBP-specific sequences from the human genome will be cloned using their affinity for MDBP to enrich for them. The distribution, frequency, and base sequences of these MDBP-specific DNA fragments will be determined. Their relative affinities for MDBP will be compared to those of high-affinity, moderate-affinity, and low-affinity sites from methylated pBR322 and M13 replicative form DNA. For several of the cloned human DNA sites, the bases involved in the interaction with MDBP will be studied by the use of DNase I, base-specific chemicals, and oligonucleotide-directed mutagenesis. By blot hybridization, in vivo transcripts of MDBP recognition sites and adjacent sequences will be sought. In addition, the effects of DNA topology and size on the binding of ligands to MDBP will be determined. Also, MDBP will be purified to apparent homogeneity and its cDNA cloned to yield an expression library for use in studying the expression of the gene(s) encoding it and, possibly, in production of functional MDBP in yeast cells. MDBP and other methylation-specific DNA-binding proteins will be sought and partially characterized in various cell populations by DNA-binding assays and, for MDBP, by the use of antibodies. These studies will help elucidate the physiological significance of MDBP and provide a test of the hypothesis that it is a repressor of transcription at certain appropriately methylated promoter sequences in the human genome.